CN101555476B - Small interfering RNA, screening method and application in preparing medicament for treating neuropathic pains - Google Patents
Small interfering RNA, screening method and application in preparing medicament for treating neuropathic pains Download PDFInfo
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- 238000012216 screening Methods 0.000 title abstract description 9
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- 208000021722 neuropathic pain Diseases 0.000 claims abstract description 18
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Abstract
The present invention discloses a small interfering RNA, a screening method and application in preparing medicament for treating neuropathic pains. The method comprises the following steps: firstly, contrasting gene sequences of EGFP and Cav2.2 e37a/37b to design a primer, constructing EGFP-e37a/37b fusion genes through a PCR method, and then screening the small interfering RNA with sequence of CAGTTTGTTGCGTTGTATTGC through interference test. The mall interfering RNA can interfere N-shaped calcium channel alpha1 subunits of pain targets, specifically interferes the expression level of an exon e37a mRNA, can generate silencing effect on e37a, but does not act on e37b. The medicament prepared by the small interfering RNA for treating the neuropathic pain has little side effect. The small interferingRNA can be used for preparing the medicament for treating the neuropathic pains (such as neuropathic headache, trigeminal neuralgia, sciatica, intercostal neuralgia, cancerous pain, phantom limb pain and the like).
Description
Technical field
The present invention relates to a kind of treatment neuropathic pain preparation technology of medicine, relate in particular to the application of a kind of siRNA and screening method and preparation treatment neuropathic pain medicine.
Background technology
N type voltage-dependent ca channel (VDCCs) has vital role in the transmission of pain and regulation and control.Their dense distribution are tip before cornu dorsale medullae spinalis nociception synapse, participates in the adjusting that main pain medium such as L-glutamic acid and P material etc. discharge.
RNA disturbs (RNA interference, RNAi) phenomenon is meant that the specificity degraded takes place the mRNA in endogenous or exogenous double-stranded RNA (dsRNA) mediated cell, cause the target gene expression silence, produce function corresponding phenotype disappearance, belong to the gene silencing mechanism after transcribing.In recent years, along with the further investigation to RNA interference phenomenon mechanism, RNAi has become a kind of effective tool of functional genome research.
Show the factor have two kinds in the Cav2.2mRNA of N type VDCCs outside, a kind of is e37a; Another kind is e37b.E37a and e37b are people, rat, the total conservative gene sequence of mouse, are 97 Nucleotide.Wherein, exon e37a is expressed in the Dorsal root ganglion neurone specifically, and is not expressed in spinal cord, oblongata, midbrain, cerebellum, thalamus, hippocampus and pallium; Another kind of exon e37b all expresses in a organized way in above-mentioned institute.
In the prior art, owing to have homology between the sequence of e37a and two exons of e37b, only there is small gap, the amino acid of these two exons codings only differs 14, therefore, the N type VDCCs blocker of clinical application is as ω-Ctx etc., when treatment pain, all can produce reticent effect to e37a and e37b.
There is following shortcoming at least in above-mentioned prior art:
Side effect is bigger, as easy inhibition sympatheticotonia, causes postural hypotension, decreased heart rate etc.; (comprising excitability and inhibitory synapse transmission) transmitted in the cynapse of inhibition maincenter, causes ataxia, dizzy, illusion, psychiatric disorder, feels sick, symptoms such as vomiting, ocular ataxy.
Summary of the invention
The purpose of this invention is to provide the application of less siRNA of a kind of side effect and screening method and preparation treatment neuropathic pain medicine.
The objective of the invention is to be achieved through the following technical solutions:
SiRNA of the present invention, the sequence of this siRNA are CAGTTTGTTGCGTTGTATTGC.
The screening method of above-mentioned siRNA of the present invention comprises step:
A, contrast EGFP reporter gene and Cav2.2e37a gene order design EGFP-e37a primer, and by this primer structure EGFP-e37a fusion gene;
Described EGFP-e37a primer comprises:
L?5’GAA?TTC?CGC?CAC?CAT?GGT?GAG?CAA?GGG?3’；
R1?5’ AGC?CAA?CGG?GTG?GCG?CAA?TAC?AAC?GCA?ACA?AAC?TGT?ACA?TAT?CCT?TAT?AATGAA?TCC?GGC?AAC?TTG?TAC?AGC?TCG?T
R2?5’AGA?TCT?TTA?CTT?GTA?GGC?CAA?CCT?ACG?AGG?GCA?GTT?CTT?CCC?TAA?GCC?AACGGG?TGG?CG?3’；
Described EGFP-e37a fusion gene comprises:
5 ' GAATTC-EGFP reporter gene-Cav2.2e37a gene-AGATCT3 ';
B, design are many to siRNA, many siRNA and described EGFP-e37a fusion gene are carried out the RNA interference test with described respectively;
C, the described RNA interference test effect of detection, the siRNA that the degree that selection is interfered described EGFP-e37a fusion gene adheres to specification.
The application of neuropathic pain medicine is treated in above-mentioned siRNA preparation of the present invention, and this siRNA is used to prepare the medicine for the treatment of neuropathic pain.
As seen from the above technical solution provided by the invention, the application of siRNA of the present invention and screening method and preparation treatment neuropathic pain medicine, owing at first contrast EGFP reporter gene and Cav2.2e37a gene order design EGFP-e37a primer, and pass through PCR, the method that enzyme is cut makes up the EGFP-e37a fusion gene, filter out the siRNA that sequence is CAGTTTGTTGCGTTGTATTGC by interference test then, this siRNA only produces reticent effect to exon e37a, with the medicine of the treatment neuropathic pain of this siRNA preparation, side effect is little.
Embodiment
SiRNA of the present invention (siRNA), its preferable embodiment is that the sequence of this siRNA is: CAGTTTGTTGCGTTGTATTGC.
The screening method of above-mentioned siRNA of the present invention, its preferable embodiment is to comprise step:
Steps A, at first, contrast EGFP reporter gene and Cav2.2e37a gene order design EGFP-e37a primer, and make up the EGFP-e37a fusion gene by this primer;
Described EGFP-e37a primer comprises:
L?5’GAA?TTC?CGC?CAC?CAT?GGT?GAG?CAA?GGG?3’；
R1?5’AGC?CAA?CGG?GTG?GCG?CAA?TAC?AAC?GCA?ACA?AAC?TGT?ACA?TAT?CCT?TAT?AATGAA?TCC?GGC?AAC?TTG?TAC?AGC?TCG?TC?3’；
R2?5’AGA?TCT?TTA?CTT?GTA?GGC?CAA?CCT?ACG?AGG?GCA?GTT?CTT?CCC?TAA?GCC?AACGGG?TGG?CG?3’；
Described EGFP-e37a fusion gene comprises:
5 ' GAATTC-EGFP reporter gene-Cav2.2 e37a gene-AGATCT 3 ';
Step B, then designs manyly to siRNA, siRNA and EGFP-e37a fusion gene are carried out RNA disturbs (RNAi) test with many respectively;
Step C, detection RNA interference test effect, the siRNA that the degree that selection is interfered the EGFP-e37a fusion gene adheres to specification.Design requirements can be for, siRNA to the jamming effectiveness of described EGFP-e37a fusion gene more than or equal to 70%, also can select other design requirements for use.
In above-mentioned RNA interference test, can also contrast the interference effect of siRNA to Cav2.2 e37b gene, specifically comprise step:
Step D, contrast EGFP reporter gene and Cav2.2 e37b gene order design EGFP-e37b primer, and by this primer structure EGFP-e37b fusion gene;
Described EGFP-e37b primer comprises:
L?5’GAA?TTC?CGC?CAC?CAT?GGT?GAG?CAA?GGG?3’；
R1?5’AAC?CCA?GAG?GTG?GGG?ACA?TGT?GTT?TCA?GCA?TCT?CAA?ACA?TGT?CAT?TGT?AACTGA?TGC?GCC?CAC?TTG?TAC?AGC?TCG?TC?3’；
R2?5’AGA?TCT?TTA?GTT?GTA?TGC?AAC?TCG?AGC?CGG?GCA?TTT?CTT?CCC?CAA?ACC?CAGAGG?TGG?GG?3’。
Described EGFP-e37b fusion gene comprises:
5 ' GAATTC-EGFP reporter gene-Cav2.2 e37b gene-AGATCT3 '.
Step e, carry out the RNA interference test with siRNA of selecting among the above-mentioned steps C and EGFP-e37b fusion gene;
Step F, detection RNA interference test effect are selected the glitch-free siRNA of EGFP-e37b fusion gene.
Above-mentioned RNA interference test can adopt following two kinds of methods:
A kind ofly be: at first, fusion gene and siRNA are put into the expression vector plasmid respectively express; Then, carry out cotransfection, carry out the RNA interference test with the expression vector plasmid of siRNA and the expression vector plasmid of fusion gene;
Another kind is: at first, with the method for liposome transfection with the integrative gene expression vector transfection to bhk cell, and the cell of selecting green fluorescence is set up the monoclonal cell strain; Then, using siRNA, is that target cell carries out the RNA interference test with the monoclonal cell strain.
Above-mentioned fusion gene can make up by the method that PCR and enzyme are cut, and after structure is finished, can also pass through one or more methods such as PCR method, enzyme blanking method, sequence measurement and identify whether constructed fusion gene sequence is correct.
The application of neuropathic pain medicine is treated in above-mentioned siRNA preparation of the present invention, and its preferable embodiment is that this siRNA is used to prepare the medicine for the treatment of neuropathic pain.The neuropathic pain here can be nervous headache, trigeminal neuralgia, sciatica, intercostal neuralgia, pain caused by cancer, phantom limb pain etc. one or more, can also be other neuropathic pain.
Specific embodiment comprises:
The gene order design primer of step 1, contrast EGFP and Cav2.2 e37a/37b, the method for cutting by PCR and enzyme has made up the EGFP-37a/37b fusion gene, by PCR, enzyme cut, method such as order-checking identifies that sequence is correct.
Step 2, according to four couples of siRNA of the principle of design (as following Tuschl rule etc.) of siRNA design, be encoded to I number, II number, III number, IV number respectively:
siRNA?I：AAGGATATGTACAGTTTGTTG；
siRNA?II：CAGTTTGTTGCGTTGTATTGC；
siRNAIII：AAGAACTGCCCTCGTAGGTTG；
siRNAIV：AACTGCCCTCGTAGGTTGGCC。
Step 3, these four couples of siRNA are carried out RNAi test with the EGFP-37a/37b fusion gene respectively.
Step 4, detect the RNAi effect with methods such as fluorescent microscope, flow cytometers: visible siRNAII obviously is suppressed the fluorescence efficiency of cell under fluorescent microscope, the RNAi efficient of the visible siRNAII of flow cytometry reached more than 80% at 48 hours, and its excess-three bar siRNA restraining effect is not obvious.And siRNAII only produces specific reticent effect to EGFP-37a;
Step 5, the effective and specificity that will obtain are disturbed the siRNA sequence of the expression level of exon e37a mRNA, use the protein expression situation of the method detection fusion gene of western blot, and the visible corresponding protein band of result obviously weakens.
Step 6, in 293FT clone, express with the full gene expression plasmid of Cav2.2, and cotransfection siRNA II expression plasmid, method with Western blot detects the protein expression situation, obtains the result consistent with step 5, thereby has verified validity and the specificity of siRNA II.
The present invention is by making up the fusion gene of EGFP reporter gene and 37a/37b, follow Tuschl Rule Design siRNA, utilize the method for fluorescent microscope and flow cytometer to detect the siRNA sequence that works, and with the method validation of western blot, promptly the protein expression situation to fusion gene and full gene detects, and obtains the siRNA sequence of the expression level of effective and specificity interference exon e37amRNA.
This siRNA sequence can be disturbed pain target spot N type calcium channel α 1 subunit, and the expression level of specificity interference exon e37amRNA, and be not only and can produce reticent effect e37a, and also inoperative to e37b, promptly e37a there is the reticent effect of specificity.
In the Dorsal root ganglion neurone of people, mouse, the expression level of exon e37a is 1/10 of exon e37b, e37a has the biophysical properties that is different from e37b, activation voltage is low and calcium current density is big, therefore though express lower but have higher biological efficiency, this provides a very significant new target drone for pain therapy.The present invention is directed to the siRNA of this target sieving, can provide new feasibility method and medicine for the neuropathic pain treatment.
The above; only for the preferable embodiment of the present invention, but protection scope of the present invention is not limited thereto, and anyly is familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement all should be encompassed within protection scope of the present invention.
Claims (3)
1. a siRNA is characterized in that, the sequence of this siRNA is CAGTTTGTTGCGTTGTATTGC.
2. the application of the described siRNA preparation of claim 1 a treatment neuropathic pain medicine is characterized in that this siRNA is used to prepare the medicine for the treatment of neuropathic pain.
3. the application of siRNA preparation treatment neuropathic pain medicine according to claim 2, it is characterized in that, described neuropathic pain comprise following one or more: nervous headache, trigeminal neuralgia, sciatica, intercostal neuralgia, pain caused by cancer, phantom limb pain.
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| CN116042712A (en) * | 2022-11-14 | 2023-05-02 | 江苏中方基因生物医学科技有限公司 | Fusion expression plasmid of novel coronavirus S protein and RFP gene and application thereof |
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| CN101060863A (en) * | 2004-10-22 | 2007-10-24 | 拉瓦勒大学 | Modulation of neuroglia-derived BDNF in the treatment and prevention of pain |
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| CN101060863A (en) * | 2004-10-22 | 2007-10-24 | 拉瓦勒大学 | Modulation of neuroglia-derived BDNF in the treatment and prevention of pain |
Non-Patent Citations (5)
| Title |
|---|
| Carsten T. Beuckmann et al.N-Type Calcium Channel alpha1B Subunit (CaV2.2) Knock-Out Mice Display Hyperactivity and Vigilance State Differences.《The Journal of Neuroscience》.2003,第 23 卷(第 17 期),6793-6797. * |
| Christophe Altier et al.Differential Role of N-Type Calcium Channel Splice Isoforms in Pain.《The Journal of Neuroscience》.2007,第 27 卷(第 24 期),6363-6373. * |
| 刘艳红.RNA 干扰技术抑制 Nav1.8 基因表达在疼痛研究中的应用.《军医进修学院博士论文 中国优秀博硕士学位论文全文数据库 (博士) 医药卫生科技辑》.2006,(第 09 期),E060-25. * |
| 卢中平等.疼痛分子治疗的研究进展.《国外医学 麻醉学与复苏分册》.2004,第 25 卷(第 2 期),71-74. * |
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